A study on L-threonine and L-serine uptake in Escherichia coli K-12

Abstract

In the current study, we report the identification and characterization of the yifK gene product as a novel amino acid carrier in E. coli K-12 cells. Both phenotypic and biochemical analyses showed that YifK acts as a permease specific to L-threonine and, to a lesser extent, L-serine. An assay of the effect of uncouplers and composition of the reaction medium on the transport activity indicates that YifK utilizes a proton motive force to energize substrate uptake. To identify the remaining threonine carriers, we screened a genomic library prepared from the yifK-mutant strain and found that brnQ acts as a multicopy suppressor of the threonine transport defect caused by yifK disruption. Our results indicate that BrnQ is directly involved in threonine uptake as a low-affinity but high-flux transporter, which forms the main entry point when the threonine concentration in the external environment reaches a toxic level. By abolishing YifK and BrnQ activity, we unmasked and quantified the threonine transport activity of the LIV-I branched chain amino acid transport system and demonstrated that LIV-I contributes significantly to total threonine uptake. However, this contribution is likely smaller than that of YifK. We also observed the serine transport activity of LIV-I, which was much lower compared with that of the dedicated SdaC carrier, indicating that LIV-I plays a minor role in the serine uptake. Overall, these findings allow us to propose a comprehensive model of the threonine/serine uptake subsystem in E. coli cells.

Keywords: Escherichia coli; L-serine uptake; L-threonine uptake; amino acid transporter; transmembrane transport.

Figure 1

Phenotype assay of the threonine-auxotrophic B1044 strain and its yifK^- derivative, B1082. The assay was performed using M9 minimal medium with 0.2% glucose and the indicated additives as described under “Phenotype assay” in the Materials and Methods. “Thr,” “Ile,” “Ser,” and “Ala-Thr” stand for L-threonine, L-isoleucine, L-serine, and L-alanyl-L-threonine, respectively. (A) Growth assay of B1044 on minimal plates with either L-threonine or L-alanyl-L-threonine as the L-threonine source. L-isoleucine was added to the medium as B1044 is unable to synthesize this amino acid along with L-threonine. L-serine was added to examine whether it inhibits L-threonine uptake. (B) Comparison of the phenotypes of B1044 and B1082 strains on minimal plates with either L-threonine or L-alanyl-L-threonine as an L-threonine source.

Figure 2

Phenotype assay of the threonine-auxotrophic B1426 strain and its derivatives carrying deletions of genes controlling threonine uptake. The assay was performed using M9 minimal medium with 0.2% glucose and the indicated additives as described under “Phenotype assay” in the Materials and Methods. “Thr,” “Ile,” “Ser,” and “Ala-Thr” abbreviations stand for L-threonine, L-isoleucine, L-serine, and L-alanyl-L-threonine, respectively. (A) Growth assay of the B1426 strain and its single, double, and triple mutant derivatives lacking the YifK, BrnQ, and LIV-I systems on minimal plates with either L-threonine or L-alanyl-L-threonine as the L-threonine source. (B) Growth assay of the B1426 strain and its double mutant derivatives possessing YifK, BrnQ, and LIV-I as the sole threonine transport system at gradually increasing L-threonine concentrations. L-isoleucine and L-serine were added as substrates competing with L-threonine for transport to examine whether they exhibit an inhibitory effect on the growth of the tested strains. (C) Phenotype assay showing the causality of BrnQ in L-threonine toxicity.

Figure 3

Assay of L-threonine transport activity. The experiments were performed as described under “Transport assay” in the Materials and Methods. The values shown are the average of three biological replicates; the shaded area and error bars indicate SD. The depicted p-values were calculated using the two-tailed Student’s t-test with unequal variances. (A) A time course of L-threonine accumulation by cells of the B2374 strain and its derivatives possessing YifK, BrnQ, or LIV-I as the sole carrier. Measurements were performed at 50 μM L-threonine. (B) Evaluation of the kinetic parameters of YifK and LIV-I carriers using L-threonine as a substrate. YifK and LIV-I transport systems were assayed using the B2396 and B2394[pMW-liv] strains, respectively. The V_max value characteristic of the LIV-I system was adjusted by dividing the value obtained for B2394[pMW-liv] cells by factor 9.85 (for more details, see the text under “Assaying the threonine uptake in the mutant strains” in the Results). The values were calculated using Lineweaver-Burk plots obtained with seven concentration points. (C) Comparison of L-threonine transport activity in B2394 strain lacking the YifK, BrnQ, and LIV-I carriers with that of B2394[pBR-brnQ] overexpressing BrnQ because of the multicopy pBR-brnQ plasmid. The vertical arrow in “↑BrnQ” indicates that BrnQ is overexpressed in this strain. The measurements were performed at 800 μM of L-threonine.

Figure 4

Characterization of the YifK carrier. The experiments were performed as described under “Transport assay” in the Materials and Methods. The values shown are the average of three biological replicates; error bars indicate SD. The depicted p-values were calculated using the two-tailed Student’s t-test with unequal variances. (A) The effect of the ion composition of a reaction mixture on the L-threonine transport activity conferred by YifK. The cells of the B2396 strain possessing YifK as a sole L-threonine carrier were grown in regular M9 medium, but the washing steps and uptake reaction were performed using M9 containing K⁺ or Na⁺ as the sole monovalent cation or a mixture of K⁺ and Na⁺ as indicated under the x-axis. For this, Na⁺-containing salts were replaced with K⁺-containing counterparts and vice versa. To obtain M9 medium with mixtures of K⁺ and Na⁺, the K⁺-based medium was supplemented with an appropriate amount of NaCl. The assay was performed at 50 μM L-threonine. The reaction time was 1 min. (B) The effect of monensin on the L-threonine transport activity of YifK. The experiments were performed using cells of the B2396 strain at 50 μM L-threonine. (C) The effect of CCCP on the L-threonine transport activity of YifK. The experiments were performed using cells of the B2396 strain at 50 μM L-threonine. (D) The effect of excessive unlabeled L-serine on the L-threonine transport activity of YifK. The experiments were performed using cells of the B2396 strain. The reaction time was 1 min. (E) The L-serine transport activity conferred by YifK. The experiments were performed using cells of the B2430 strain lacking all known L-serine carriers and its ∆yifK derivative B2429 strain at the indicated L-serine concentrations and a reaction time of 1 min.

Figure 5

Involvement of the LIV-I carrier in L-serine uptake. The experiments were performed as described under “Transport assay” in the Materials and Methods. The velocity of uptake was measured at 100 μM L-serine. The reaction time was 1 min. The vertical arrow in “↑LIV-I” indicates that LIV-I is overexpressed in strains harboring the pMW-liv plasmid. The values shown are the average of three biological replicates; error bars indicate SD. The depicted p-values were calculated using the two-tailed Student’s t-test with unequal variances.

Figure 6

Schematic of the L-serine and L-threonine uptake routes in E. coli K-12 cells. Dotted and solid lines represent the outer and cytoplasmic membranes, respectively. Amino acid designations under braces indicate the substrate specificity of indicated transport systems. Green highlighting indicates the transport systems first discovered and reported in the present work (YifK) or characterized as carriers capable of translocating threonine (LIV-I and BrnQ). Previously known serine/threonine carriers (SdaC, TdcC, and SstT) are highlighted in blue. For more details, see the Discussion.